This Application is a 371 of PCT/EP98/04723 filed on Jul. 27, 1998, which claims benefit of German Application 197 32 829.6 filed on Jul. 30, 1997.
The present invention relates to processes for producing carrier-bound proteins, in particular S-layer proteins and modified S-layer proteins in pro- or eukaryotic host cells.
Crystalline bacterial cell surface layers (S-layers) form in many eubacteria and in most archaebacteria of the outermost cell wall component (Sleytr et al. (1988), Crystalline Bacterial Cell Surface Layers, Springer Verlag Berlin; Messner and Sleytr. Adv. Mikrob. Physiol. 33 (1992), 213-275). Most of the S-layer proteins known at present are composed of identical proteins or glycoproteins which have apparent molecular weights in the range from 40,000 to 220,000. The components of S-layers are self-assembling and most lattices have oblique (p2), square (p4) or hexagonal (p6) symmetry. The functions of bacterial S-layers are still not completely known but, on the basis of their localization on the cell surface, it is likely that the porous crystalline S-layers act mainly as protective coverings, molecular sieves or for promoting cell adhesion and surface recognition.
Genetic data and sequence information are known for various S-layer genes from microorganisms. A review is to be found in Peyret et al., Mol. Microbiol. 9 (1993), 97-109. Express reference is made to these data. The sequence of the gene sbsA coding for the S-layer protein of B.stearothermophilus PV72 and a method for cloning it are indicated by Kuen et al. (Gene 145 (1994), 115-20). B.stearothermophilus PV72 is a Gram-positive bacterium which is covered by a hexagonally arranged S-layer. The main component of the S-layer is a 128 kd protein which is the commonest protein in the cell, comprising approximately 15% of the total protein constituents. Various strains of B.stearothermophilus have been characterized and differ in the type of, S-layer lattice, the molecular weight and the glycosylation of the S-layer components (Messner and Sleytr (1992), supra).
German Patent Application DE-A 44 25 527 discloses the signal peptide-encoding section of the S-layer gene of B.stearothermophilus and the amino acid sequence derived therefrom. The cleavage site between the signal peptide and the mature protein is located between position 30 and 31 of the amino acid sequence. The signal peptide-encoding nucleic acid can be operatively linked to a protein-encoding nucleic acid and used for the recombinant production of proteins in a process in which a transformed host cell is prepared, the host cell is cultivated under conditions which lead to expression of the nucleic acid and to production and secretion of the polypeptide encoded thereby, and the resulting polypeptide is isolated from the culture medium. The host cells mentioned as preferred are prokaryotic organisms, in particular Gram-positive organisms of the genus Bacillus.
The international Patent Application PCT/EP97/00432 proposes the recombinant production of S-layer proteins and modified S-layer proteins in the cytoplasm of Gram-negative host cells.
It has now been found, surprisingly, not only that the recombinant production of S-layer proteins is possible in the cytoplasm of Gram-negative prokaryotic host cells, but also that recombinant expression comprising integration in the outer or the cytoplasmic membrane, secretion into the periplasm or/and secretion into the extracellular space can be carried out. It has additionally been found that recombinant expression of S-layer proteins also takes place in the eukaryotic host cells.
A first aspect of the present invention is thus a process for producing S-layer proteins, which comprises
(a) preparing a Gram-negative prokaryotic host cell which is transformed with a nucleic acid which codes for an S-layer protein and is operatively linked to a signal sequence which codes for a peptide which brings about integration of the S-layer protein in the outer membrane of the host cell, integration of the S-layer protein in the cytoplasmic membrane of the host cell, secretion of the S-layer protein into the periplasmic space of the host cell or/and secretion into the medium surrounding the host cell,
(b) cultivating the host cell under conditions leading to expression of the nucleic acid and to production of the polypeptide encoded thereby, and
(c) where appropriate isolating the resulting polypeptide from the outer membrane of the host cell, from the cytoplasmic membrane of the host cell from the periplasmic space of the host cell or/and from the medium surrounding the host cell.
A second aspect of the present invention is a process for producing S-layer proteins, which comprises
(a) preparing a eukaryotic host cell which is transformed with a nucleic acid which codes for an S-layer protein and is preferably operatively linked to a signal sequence which brings about integration of the S-layer protein in the cytoplasmic membrane of the host cell, integration of the S-layer protein into an organelle of the host cell or/and secretion into the medium surrounding the host cell,
(b) cultivating the host cell under conditions leading to expression of the nucleic acid and to production of the polypeptide encoded thereby, and
(c) where appropriate isolating the resulting polypeptide from the cytoplasmic membrane of the host cell, from an organelle of the host cell or/and from the medium surrounding the host cell.
It has been found, surprisingly, that secretion of any heterologous S-layer proteins, including recombinant S-layer proteins, into the periplasmic space of a Gram-negative host cell or even secretion into the medium surrounding the host cell is possible. This entails the S-layer protein being formed in the periplasm of the host cell not in the form of unordered inclusion bodies but, unexpectedly, in the form of ordered monomolecular layers. In addition, anchoring of heterologous S-layer proteins in the outer or the cytoplasmic membrane of Gram-negative host cells is possible.
S-layer proteins can also be expressed in functional form in eukaryotic cells such as, for example, mammalian cells or yeast. Glycosylation takes place in the case of recombinant S-layer proteins having a eukaryotic fusion portion. In addition, glcosylation may take place in the S-layer protein portion itself.
The process according to the invention makes it possible preferably to express S-layer genes derived from B.stearothermophilus PV72,in particular to express the S-layer genes sbsA and sbsB. In addition, however, it is also possible to express S-layer genes from other organisms (cf., for example, Peyret et al., (1993) supra) by the process according to the invention.
The nucleotide sequence of the gene coding for the mature SbsA protein is indicated in SEQ ID NO. 1 from position 91-3684. The relevant amino acid sequence is depicted in SEQ ID NO. 2. The nucleotide sequence for the gene coding for the mature SbsB protein is indicated in SEQ ID NO. 5 from position 94-2763. The a relevant amino acid sequence is depicted in SEQ ID NO. 6.
In a first preferred embodiment (sbsA), the nucleic acid coding for an S-layer protein is selected from
(i) a nucleic acid which comprises the nucleotide sequence shown in SEQ ID NO. 1 from position 91 to 3684,
(ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the scope of the degeneracy of the genetic code, and
(iii) a nucleic acid which comprises a nucleotide sequence hybridizing with the nucleic acids from (i) or/and (ii) under stringent conditions.
In a second preferred embodiment (sbsB), the nucleic acid coding for an S-layer protein is selected from
(i) a nucleic acid which comprises the nucleotide sequence shown in SEQ ID NO. 5 from position 94 to 2763,
(ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the scope of the degeneracy of the genetic code, and
(iii) a nucleic acid which comprises a nucleotide sequence hybridizing with the nucleic acids from (i) or/and (ii) under stringent conditions.
The term xe2x80x9cstringent hybridizationxe2x80x9d means for the purpose of the present invention that hybridization still occurs even after washing at 55xc2x0 C., preferably 60xc2x0 C., in an aqueous low-salt buffer (for example 0.2xc3x97SSC) (see also Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual.
Gram-negative prokaryotic host cells are used in the first aspect of the invention. In this case, surprisingly, an S-layer protein assembled in an ordered structure is obtained in the periplasm. The host cells preferably used are enterobacteria, in particular E.coli. Examples of suitable E.coli strains are DH5xcex1 (sup E44, xcex94 lac U169, hsdR17, recA1, endA1, gyr A96, thi-1, rel A1; Hanahan, J. Mol. Biol. 166 (1983), 557-580) and HB 2151 (K12, ara. xcex94 (lac-pro), thi/Fxe2x80x2, pro A+B+, laclqZxcex94M15; Pharmacia Biotech).
Eukaryotic host cells are used in the second aspect of the invention. Yeast cells, mammalian cells such as, for example, CHO cells or human cells, insect cells or plant cells are preferably used.
The process according to the invention can also be employed for obtaining recombinant S-layer proteins. This is done by using a nucleic acid coding for the S-layer protein and comprising one or more insertions which code for peptide or polypeptide sequences. These insertions may, on the one hand, code only for peptides with a few amino acids, for example 1-25 amino acids. On the other hand, the insertions may also code for larger polypeptides of, for example, up to 1000 amino acids and preferably up to 500 amino acids, without the S-layer protein losing the ability to form a correctly folded structure. Besides the insertions, the recombinant S-layer protein may also comprise amino acid substitutions, in particular substitutions of single amino acids in the region of the insertion site, and, where appropriate, deletions of single amino acids or short amino acid sections of up to 30 amino acids.
Preferred insertion sites for peptide- or polypeptide-encoding sequences in the sbsA gene are regions between positions 200-3600 of the nucleotide sequence shown in SEQ ID NO. 1. Particularly preferred insertion sites are the NruI cleavage site at position 585,the PvuII cleavage site at position 881,the SnaB I cleavage site at position 920,the PvuII cleavage site at position 2507 and the PvuII cleavage site at position 2652 (PCT/EP 97/00 432). Further preferred insertion sites are positions 562, 1087, 1813, 1947, 2295, 2652, 3046, 3484 and 3594. The positions stated in each case relate to the first nucleotide of the insertion.
Preferred insertion sites into the sbsB gene are regions between positions 200-2600 of the nucleotide sequence shown in SEQ ID NO. 5. Particularly preferred insertion sites are positions 410 (codon 136), 484 (codon 161/162) and 1583 (codon 528/529) (PCT/EP 97/00432). Further preferred insertion sites are positions 598, 1012, 1435, 1808 and 2301,the position indicated in each case relating to the first nucleotide of the insertion.
The peptide- or polypeptide-encoding insertions are preferably selected from nucleotide sequences which code for cysteine residues, regions with several charged amino acids, for example Arg, Lys, Asp or Glu, or Tyr residues, DNA-binding epitopes, antigenic, allergenic or immunogenic epitopes, metal-binding epitopes, streptavidin, enzymes, cytokines or antibody-binding proteins.
A particularly preferred example of an insertion into the nucleic acid coding for the S-layer protein is a nucleotide sequence coding for streptavidin. It is possible in this way to obtain universal carrier molecules which are suitable for coupling biotinylated reagents to the integrated streptavidin of the recombinant S-layer protein and for detection in immunological or hybridization test methods.
Another preferred example of insertions comprises antigenic, allergenic or immunogenic epitopes, for example epitopes from pathogenic microorganisms such as, for example, bacteria, fungi, parasites etc. and viruses, or epitopes from plants or epitopes against endogenous substances, for example cytokines, and against toxins, in particular endotoxins. Particularly preferred examples of immunogenic epitopes are epitopes from viruses, for example from herpesviruses such as, for example herpesvirus 1,for example glycoprotein xcex94, herpesvirus 6 or pseudorabiesvirus (Lomniczi et al., J. Virol. 49 (1984), 970-979), in particular epitopes from the gB, gC or/and gD genes, epitopes from foot and mouth disease virus (FMDV), in particular epitopes from the gene sections which code for VP1, VP2 or/and VP3, epitopes from flaviviruses or epitopes from filoviruses such as, for example, Ebola, Marburg or Lassa virus. The immunogenic epitopes can be selected so that they promote the generation of an antibody-mediated immune response or/and promote the generation of a cellular immune response, for example by stimulating T cells. Examples of suitable allergenic epitopes are birch pollen allergens, for example Bet v I (Ebner et al., J. Immunol. 150 (1993) 1047-1054). Also particularly preferred are antigenic epitopes able to bind and filter out, from serum or other body fluids, endogenous or exogenous substances such as, for example, cytokines or toxins. Epitopes of this type may comprise constituents of cytokine receptors or toxin receptors or of antibodies against cytokines or toxins.
Modified S-layer proteins comprising immunogenic or/and antigenic epitopes with glycosylation sites are preferably produced in eukaryotic host cells in which glycosylation is possible. It is also possible in this case for the natural S-layer sequences to be glycosylated. Examples of potential N-glycosylation sites in the S-layer gene sbsA are amino acid positions 26, 285, 343, 387, 388, 418, 421, 483, 653, 675, 902, 924, 1048, 1058, 1118, 1154 and 1161. A potential N-glycosylation may take place in the sbsB gene at positions 155, 184, 213, 302, 303, 400, 463, 606, 755 and 915. Further possible modifications of the sbsA gene comprise amidation, phosphorylation by casein kinase II, N-myristoylation and phosphorylation by protein kinase C. Further possible modifications of the sbsB gene comprise phosphorylation by cAMP- and cGMP-dependent protein kinase, phosphorylation by casein kinase II, N-myristoylation, phosphorylation by protein kinase C and attachment to a fibronectin receptor (via sequence RGD).
On the other hand, the insertions may also code for enymzes. Preferred examples are enzymes for synthe-sizing polyhydroxybutyric acid, for example PHB synthase. Incorporation of PHB synthase into the S-layer may produce, on addition of the substrate hydroxybutyric acid under suitable conditions, a molecular spinneret. Another preferred example of an enzyme is bacterial luciferase. In this case, a molecular laser can be obtained on addition of the enzyme substrate, an aldehyde, and FMNH2 (reduced flavin mononucleotide), and in the presence of O2.
There is likewise preference for insertions which code for cytokines such as, for example, interleukins, interferons or tumor necrosis factors. These molecules can be used, for example, in combination with immunogenic epitopes for producing vaccines.
Finally, there is also preference for insertions which code for antibody-binding proteins such as, for example, protein A or protein G or for DNA- or/and metal-binding epitopes such as, for example, leucine zippers, zinc fingers etc.
Thus, the present invention provides for the first time a Gram-negative prokaryotic cell which comprises immobilized recombinant polypeptides in native form, for example active enzymes, in the outer membrane, in the cytoplasmic membrane, preferably on the inside thereof or/and in the periplasm. It is possible in this way for 50,000-200,000,for example about 100,000, recombinant molecules to be immobilized per mm2 of recombinant S-layer. Up to 3000 m2 of S-layer can be obtained per kg of recombinant E.coli cells.
The present invention further provides for the first time a eukaryotic cell which comprises immobilized recombinant S-layer polypeptides in the cytoplasmic membrane, preferably on the inside thereof or/and in cell organelles such as, for example, the Golgi apparatus, lysosomes, mitochondria, chloroplasts, vacuoles or endoplasmic reticulum.
Preference is further given, in particular for secretion into the periplasm, to recombinant S-layer proteins into which cysteine residues have been incorporated. It is possible, by a selection of the insertion positions, to achieve covalent crosslinking of the S-layers in the periplasm or/and on insertion at positions unsuitable for crosslinking it is possible to provide docking sites for polypeptides, for example for enzymes, which can be covalently linked via a free SH group to the S-layer. Suitable and particularly preferred for this purpose are recombinant polypeptides into which an additional cysteine residue has been introduced by genetic manipulation methods, preferably at the N or C terminus or at a domain localized on the surface and which, through selection of a suitable expression system, are likewise secreted into the periplasm of the recombinant host cell.
In the process according to the invention, the nucleic acid coding for the S-layer protein is used operatively linked to a nucleic acid coding for a signal peptide of Gram-negative bacteria or of eukaryotic cells, i.e. the signal peptide-encoding nucleic acid is located on the 5xe2x80x2 side of the S-layer protein-encoding nucleic acid.
On integration into the outer membrane of prokaryotic Gram-negative host cells, the C-terminal domain of the IgA protease from neisseria or haemophilus (Klauser et al., J. Mor Bio. 234 (1993), 579-593) can be used as signal peptide-encoding sequence.
On integration into the cytoplasmic membrane of Gram-negative prokaryotic host cells it is preferable to use a hydrophobic membrane-integrating protein domain which has no lytic activity and has an xcex1-helical structure. Examples of DNA sequences which code for such a membrane-integrating protein domain are described in European patent 0 516 655.
On secretion into the periplasm of Gram-negative prokaryotic cells it is possible for the nucleic acid coding for the signal peptide to comprise (a) the signal peptide-encoding section of the nucleotide sequence depicted in SEQ ID NO. 7 and FIG. 4, (b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code or/and (c) a nucleotide sequence which is at least 80% and, in particular, at least 90% homologous with the sequences from (a) or/and (b). Other sequences which bring about secretion into the periplasm are described, for example, by Blondel and Bedouelle (Eur. J. Biochem 193 (1990), 325-330; Adip-Conquy et al. (Protein Eng. 8 (1995), 859-863); Weller et al (Eur. J. Biochem. 236 (1996), 34-39) and Dubreuil et al. (FEMA Immunol. Med. Microbiol. 13 (1996), 317-323).
On secretion into the extracellular medium of Gram-negative prokaryotic cells it is possible for the nucleic acid coding for the signal peptide to comprise (a) the signal peptide-encoding section of the nucleotide sequence depicted in SEQ ID NO. 8 and FIG. 5, (b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code or/and (c) a nucleotide sequence which is at least 80% and, in particular, at least 90% homologous with the sequences from (a) or/and (b). However, other signal peptide-encoding sequences are also suitable in addition, as described, for example, by Yuan et al. (Appl. Environ. Microbiol. 63 (1997), 263-269) and Hoogenboom et al. (Nucleic Acids Res. 19 (1991), 4133-4137).
Signal peptide-encoding nucleic acids known for expression in the cytoplasmic membrane or in organelles of eukaryotic cells are the N-terminal transit peptide of plastocyanin for transport in chloroplasts (Weisbeek et al., J. Cell. Sci. Suppl. 11 (1989), 199-223), mitochondrial signal peptides for transport in mitochondria (Skerjanc, Biochem. Cell. Biol. 68 (1990), 9-16), targeting sequences for transport in vacuoles (Vitale and Chrispeels, Bioessays 14 (1992), 151-160), targeting sequences for the cell membrane, the cytoplasm and the Golgi apparatus (Stanley, Mol. Membr. Biol. 13 (1996), 19-27), retention signals for the endoplasmic reticulum (Lencer et al., J. Cell. Biol. 131 (1995), 951-962) and transfer sequences for the Golgi apparatus or the plasma membrane (Rambourg et al., Anat. Rec. 245 (1996), 447-458).
Signal peptide-encoding nucleic acids known for secretion into the extracellular medium of eukaryotic cells are the hsp 150 delta carrier (Jamsa et al., Yeast 11 (1995), 1381-1391), the signal peptide of melittin from the honeybee (Sisk et al., J. Virol 68 (1994), 766-775), signal peptides from baculovirus (Murphy et al., Protein Expr. Purif. 4 (1993), 349-357), fragments of the K1 killer preprotoxin (Cartwright et al., Yeast 8 (1992), 261-272), the signal peptide and the N-terminal proregion of peptidylglycine xcex1-hydroxylating monooxygenase (Mains et al., Mol. Endocrinol. 9 (1995), 3-13), the maltose-binding protein MalE with its signal sequence (Staropoli et al., J. Virol. Methods 56 (1996), 179-189;Clement and Jehanna, J. Biotechnol. 43 (1995), 169-181), the prepro-xcex1-factor leader region of the yeast MF xcex11 gene (Elliot et al., Gene 79 (1989), 167-180), the signal sequence of the IL-1 receptor antagonist (Wingren et al., Cell Immunol. 169 (1996), 226-237), the signal peptide of the wheat xcex1-amylase gene (Ribbe and Nagarajan, Mol. Gen. Genet. 235 (1992), 333-339), secretion polypeptides from fungi (Punt et al., Antonio Van Leeuwenhoek 65 (1994), 211-216), the leader peptide of the killer toxin from Kluyveromyces lactis (Baldari et al., EMBO J. 6 (1987), 229-234) and the inulinase signal sequence (Kang et al., J. Biotechnol. 48 (1996), 15-24). Fusion constructs from MalE and SbsA and from MalE and SbsB are described in the present application.
Besides the section coding for the signal peptide, the DNA sequence coding for the S-layer protein may comprise one or more other sections which code for other protein domains. Such a section may preferably be located between the section coding for the signal peptide and the section coding for the S-layer protein. This section preferably codes for a secretory polypeptide from Gram-negative bacteria or eukaryotic organisms or a part thereof. A preferred example of such a nucleic acid section is the malE gene which encodes the maltose-binding protein.
In a preferred embodiment of the process according to the invention, it is also possible to express several S-layer genes in a single host cell. For this purpose there is preferably expression of at least two S-layer genes, in which case one bf them codes for a modified S-layer protein and another codes for an unmodified S-layer protein. The unmodified S-layer protein is preferably able to form an S-layer structure which is compatible with the modified S-layer protein. One example of this embodiment of the process according to the invention is an E.coli cell which is transformed with two S-layer genes, one of which is a natural sbsA or sbsB gene and the other is a recombinant sbsA or sbsB gene.
The present invention further relates to a nucleic acid which codes for an S-layer protein optionally comprising heterologous peptide or polypeptide insertions and is operatively linked to a signal sequence which codes for a peptide which brings about
(a) integration into the outer or cytoplasmic membrane of a Gram-negative prokaryotic host cell, secretion into the periplasmic space of a Gram-negative prokaryotic host cell or/and secretion into the extra-cellular medium of a Gram-negative prokaryotic host cell, or
(b) integration into the cytoplasmic membrane of a eukaryotic host cell, integration into an organelle of a eukaryotic host cell or/and secretion into the extracellular medium of a eukaryotic host cell.
The nucleic acid preferably codes for a recombinant S-layer protein as indicated above.
The present invention further relates to a recombinant vector which comprises at least one copy of a nucleic acid according to the invention. The vector is preferably replicable in prokaryotes or/and in eukaryotes. The vector is particularly preferably a prokaryotic or eukaryotic plasmid. It is further preferred for the vector to be the nucleic acid according to the invention operatively linked to an expression control sequence which is active in Gram-negative or eukaryotic cells. The expression control sequence particularly preferably comprises a regulable promoter. Examples of suitable prokaryotic promoters are the tac, lac, trp or xcex promoter. Examples of suitable eukaryotic promoters are the SV40, CMV or metallothionein promoter.
The present invention further relates also to a host cell which is transformed with a nucleic acid or a recombinant vector according to the present invention. The cell is preferably a Gram-negative prokaryotic cell, for example an E.coli cell, or a eukaryotic cell, for example a yeast cell or a CHO cell. The cell according to the invention may comprise a recombinant S-layer structure in the cytoplasmic membrane, the periplasm or a cell organelle. Processes for the transformation of cells with nucleic acids are general prior art (see Sambrook et al., supra) and therefore need not be explained.
A recombinant S-layer structure comprising as subunit at least one recombinant S-layer protein according to the invention can be assembled from recombinant S-layer protein molecules. It is further preferred for the S-layer structure according to the invention also to comprise unmodified S-layer proteins as xe2x80x9cdiluting moleculesxe2x80x9d. The unmodified S-layer proteins are preferably present in a molar proportion of 10-99% based on the total S-layer proteins.
The S-layer structure according to the invention may comprise several layers which are linked together covalently or by affinity binding. Covalent linkages can be introduced, for example, by insertions of cysteine residues and a subsequent formation of cystine bridges. Linkages by affinity binding comprise, for example, antibody-antigen, antibody-protein A or antibody-protein G or streptavidin-biotin interactions.
S-layer structures comprising recombinant S-layer proteins may also be produced where appropriate in carrier-bound form. This can be done by reassembling the S-layer structure from individual units in the presence of a peptidoglycan carrier, producing, for example, peptidoglycan layers which are covered on one or both sides with an S-layer structure. Another possibility for producing carrier-bound S-layer structures is to produce a layer of S-layers at an interface between two media, for example water/air, and to immobilize this layer on a solid phase, for example a filter membrane (cf., for example, Pum and Sleytr (1994), Thin Solid Films 244, 882-886; Kxc3xcpcxc3xc et al. (1995), Biochim. Biophys. Acta 1235, 263-269).
The recombinant S-layer proteins and S-layer structures are suitable for a large number of applications. A particularly preferred use is as vaccine or adjuvant, in which case the recombinant S-layer proteins used comprise immunogenic epitopes of pathogens and/or endogenous immunostimulant polypeptides such as, for example, cytokines. Purification of the recombinant S-layer proteins is not absolutely necessary for this application. It is possible instead to use, for example, a combination with a bacterial ghost which comprises additional immunogenic epitopes where appropriate in its periplasmic space, its outer membrane or its cytoplasmic membrane.
The production of suitable xe2x80x9cbacterial ghostsxe2x80x9d is described, for example, in the International Patent Application PCT/EP91/00967, to which reference is made herewith. This discloses modified bacteria obtainable by transformation of a Gram-negative bacterium with the gene of a membrane protein having lytic activity from bacteriophages, with the gene of a toxin-release protein having lytic activity or with genes which comprise part-sequences thereof which code for lytic proteins, cultivation of the bacterium, expression of this lysis gene and isolation of the resulting bacterial ghost from the culture medium.
A recombinant protein which is obtainable by expression of a recombinant DNA in these Gram-negative bacteria can be bound to the membrane of these bacteria as described in European Patent 0 516 655. This recombinant DNA comprises a first DNA sequence which codes for a hydrophobic membrane-integrating protein domain which has no lytic activity, has an xcex1-helical structure and consists of 14-20 amino acids which may be flanked N- and C-terminally by, in each case, 2-30 suitable amino acids. A second DNA sequence which codes for a required recombinant protein is operatively linked to this first DNA sequence. The Gram-negative bacterium additionally comprises a third DNA sequence which is subject to a control separate from the first and second DNA sequences and codes for a membrane protein having lytic activity from bacteriophages or a toxin-release protein having lytic activity or for the parts thereof having lytic activity. So-called xe2x80x9cbacterial ghostsxe2x80x9d are obtained by expression and lysis of such recombinant Gram-negative bacteria and comprise an intact surface structure with immunogenic epitopes bound to the surface.
On combination of these bacterial ghosts with recombinant S-layers according to the invention it is possible to produce vaccines and adjuvants which have particularly advantageous properties.
Another particularly preferred use of recombinant S-layer proteins and S-layer structures is the use as enzyme reactor. Such an enzyme reactor can be formed, for example, by a cell which comprises in its interior a recombinant S-layer structure according to the invention. On the other hand, the enzyme reactor may also be formed from isolated S-layer structures which have been reassembled in vitro, or combinations of various S-layer structures.